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Advanced approaches to the synthesis and reconstruction of genetic material developed in the Institutes of Molecular Biology and Genetics during the past years are summarized. The evolution of methods for oligonucleotide synthesis and scopes for their use in gene production are discussed. The principles of localised mutagenesis methods developed in the Institute are described, such as: a) mutagenesis directed to the regulatory gene regions; b) segment-localized mutagenesis; c) mutagenesis directed by phosphotriester analogues of oligonucleotides. Examples of employing these methods for induction of regulatory mutants of phage lambda, production of fused genes, mutant interferon genes, construction of new DNA vectors, construction of hybrid H1-H3 subtype haemagglutinine gene of influenza virus etc. are presented. The approach to in vivo site-directed mutagenesis is experimentally substantiated. 相似文献
3.
L P Kravchenko A N Andrienko A Iu Petrenko A N Sukach 《Ukrainski? biokhimicheski? zhurnal》1989,61(4):112-116
A method for isolating hepatocytes using the two-step liver perfusion by chelate-containing solutions is described. The hepatocytes obtained are highly inactive for the trypan blue staining test, (not less than 90%), ATP content, the rate of endogenic respiration and its stimulation by succinate, a degree of xenobiotic oxidation. 相似文献
4.
Slow centrifugation of isotonic sucrose medium containing the suspension of hepatocytes, obtained after disaggregation of liver, which has been preliminarily perfused by EDTA solution, results in the formation of two fractions of cells of different colour. Fraction 1 contains intact and metabolically active hepatocytes. Cells of fraction 2 have a damaged membrane, but they are capable of realizing biotransformation of xenobiotics in the presence of exogenous NADPH as well as maintaining efficiency of oxidative phosphorylation of mitochondria under the transfer to the medium simulating cytoplasm. 相似文献
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V V Smirnov E L Mishenkova Iu A Grinevich G T Petrenko S V Martynenko 《Antibiotiki i khimioterapii͡a》1990,35(6):31-34
It was shown that asterin therapy performed on a model of spontaneous metastasis markedly changed the content of the thymus serum factor in mice and normalized its level. The inducing mechanism of the drug's antitumor action was realized both at the stage of the metastatic postinvasion phase and during its terminal period which was probably due to an increase in the influence of the thymus on the immune system and evident from an increase in the therapeutic action of the drug. 相似文献
7.
A G Petrenko V A Kovalenko O G Shamotienko I N Surkova T A Tarasyuk Ushkaryov YuA E V Grishin 《The EMBO journal》1990,9(6):2023-2027
The receptor protein of alpha-latrotoxin (alpha LTx, a neurotoxin with 'pure' presynaptic action isolated from black widow spider venom), was solubilized by Triton X-100 from bovine brain membranes and purified by affinity chromatography on alpha LTx-Sepharose. The purified receptor preparation contained four major polypeptides of molecular masses 200 (alpha), 160 (alpha'), 79 (beta) and 43 (gamma) kd according to SDS electrophoresis with molecular ratio alpha 1 alpha' 1 beta 2 gamma 2. The alpha- and alpha'-subunits are glycoproteins binding to wheat germ lectin and can be separated under non-denaturing conditions by anion exchange chromatography. Purified to homogeneity, both of them, though differing in the carbohydrate composition, retain the alpha LTx-binding activity and give closely related peptide maps. Anti-alpha antibodies recognize the alpha'-subunit as well. These results suggest that alpha LTx receptor is present in purified preparations in two very close forms containing the alpha- or alpha'-subunit. Beta and gamma proteins do not specifically bind alpha LTx and their physiological role is unclear. They form a complex with solubilized alpha- and alpha'-subunits independently of alpha LTx presence. The receptor proteins were purified to homogeneity by high performance gel filtration in the presence of SDS, their amino acid composition was determined. 相似文献
8.
This study deals with isolation of rat hepatocytes by a non-enzymatic method and the separation of intact and damaged cells in sucrose medium. Low speed centrifugation in isotonic sucrose medium of a hepatocyte suspension obtained by mechanical desaggregation of liver pre-perfused with EDTA solution results in the formation of a cell pellet which contains two different layers. A darker layer contains hepatocytes with intact plasma membranes. Their respiratory activity and xenobiotic metabolism are close to those of the cells isolated by collagenase perfusion. The study of distribution of lipophilic cation tetraphenylphosphonium (TPP+) indicates a predominantly mitochondrial localization of TPP+ in the intact cells following non-enzymatic and collagenase isolation. Hepatocytes in the upper layer have damaged plasma membranes. As a result they lose the potential to accumulate TPP+, and have low rates of endogenous respiration and biotransformation activity. Addition of exogenous NADPH restores the capability to metabolize xenobiotics. Washing and incubation of these hepaticytes in an intracellular type medium results in restoration of uncoupler-stimulated oxygen consumption and generation of membrane potential in the presence of a succinate substrate. These properties are close to those of hepatocytes permeabilized by digitonin treatment. Thus, the procedure allows the simultaneous isolation of both intact and permeabilized hepatocytes with functionally active intracellular structures without the use of relatively expensive chemicals such as collagenase and Percoll.Abbreviations 4-OHBP
4-hydroxybiphenyl
- BP
biphenyl
- BSA
bovine serum albumin
- DNP
2,4-dinitrophenol
- EDTA
ethylendiamintetraacetate
- NADPH
nicotinamide adenine dinucleotide phosphate reduced
-
p-NA
p-nitroanisole
-
p-NPh
p-nitrophenol
- TPP+
tetraphenylphosphonium 相似文献
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I. V. Sorokina D. S. Baev N. A. Zhukova T. G. Tolstikova A. N. Antimonova N. I. Petrenko E. E. Shults I. A. Grigor’ev 《Russian Journal of Bioorganic Chemistry》2013,39(6):668-670
Betulonic acid amides containing the nitroxyl radical moiety exert an anticholestatic effect in mice. The introduction of piperidine nitroxide moiety into the lupane core increases its hepatoprotective activity. Oral administration of the piperidine nitroxide derivative in a dose of 50 mg/kg does not stimulate transplanted tumor growth and increases lifespan in mice. 相似文献